DESCRIPTION: This is a renewal application to continue studies on chromosome replication in Saccharomyces. The goal is to provide a detailed understanding of the patterns of chromosomal replication, that is, what is the distribution of replication origins on a eukaryotic chromosome, what is the usage frequency of these origins, what is the temporal pattern of activation of these origins, are there upper limits to the size of the chromosomal region replicated from a particular origin, are there specific termination sites for replication or is this a passive process, what are the sequence/structure characteristics of a replication origin, what role is played by the chromosomal environment and what are the underlying mechanisms controlling these patterns? During the previous grant periods, Dr. Newlon and coworkers succeeded in mapping the origins of replication of Saccharomyces cerevisiae chromosome III. Only a small region of the right arm of chromosome III distal to MAT remains to be studied and completion of this aspect of the project is proposed in this application. The work demonstrated that sequences that function as replication origins are dependent upon the ARS (autonomously replicating sequence) located at that site. ARS sequences are identified as DNA fragments which stabilize the retention of episomal plasmids. It has yet to be determined if sequences lacking ARS activity are able to function as origins. Dr. Newlon plans a structure-function analysis of ARS307 and 309. These were chosen because both are highly active origins used in nearly 100% of the division cycles and because deletion of both origins together increases the rate of chromosome loss 50-200 fold. Using site-directed mutagenesis, she will determine the essential sequence elements of these ARSs and relate her results to the newly expanded ARS consensus sequence. ARS307 has a linked stimulator sequence which maps outside of the core consensus elements. Dr. Newlon will identify this stimulator and any interacting proteins. ARS309, while active within chromosomal regions, is inactivated when placed close to a telomere. This affect is a function of the sequences flanking ARS309 and Dr. Newlon will explore the basis of this deleterious effect with particular interest in assessing the role of genes known to affect telomere silencing such as the SIR genes. Dr. Newlon is interested in the trans-acting functions which act at the ARS element and function during replication initiation or give the various ARS elements functionally distinct characteristic. Toward this goal, she will identify proteins which interact with ARS307 and ARS309 using what she calls a "one-hybrid system". Additionally, members of the CDC46 family, which include MCM2, MCM3 and CDC 46,47 and 54); the MCM class is known to affect mini-chromosome stability will be tested. Dr. Newlon plans to determine the effect on replication origin usage in cells depleted of either the CDC46, 47 or 54 gene product. In the course of an analysis of the effects of deleting origins, Dr. Newlon discovered that a chromosome III derivative which lacked all 6 of its active origins exhibited only a 3-4 fold decrease in stability so long as it is paired with an homologous S. cerevisiae chromosome III. Its stability is decreased about 36-fold if a S. carlsbergensis partner is present instead. Dr. Newlon will investigate the mechanism of this homology-dependent stabilization.